| Objective:To investigate the role of Nur77 in regulating NETosis in lung injury induced by lipopolysaccharide（LPS）in mice with acute respiratory distress syndrome（ARDS）.Methods:（1）Sixteen Wild Type（WT）C57BL/6J mice and 16 Nur77 knockout(Nur77-/-)mice were assigned to 4 groups according to the principle of random block design.There were 8 mice in each group,including WT+NS group,WT+LPS group,Nur77-/-+NS group,and Nur77-/-+LPS group.The severe ARDS mouse model was constructed by intratracheal infusion of LPS（20mg/kg）.The survival time of mice was observed for 72 hours,and the survival curve was drawn.（2）Thirty-six wild type（WT）C57BL/6J mice and 36 Nur77-/-mice were randomly assigned into four groups:WT+NS group,WT+LPS group,Nur77-/-+NS group and Nur77-/-+LPS group.Design 4 h,12 h,24 h,a total of 3 time points,each group each time point 6.The ARDS mouse model was established by endotracheal intubation with LPS（5mg/kg）and the control group was established by endotracheal intubation with normal saline（NS）.The mice were killed by orbital bloodlet at 4 h,12 h and 24 h after modeling,and lung tissue and bronchoalveolar lavage fluid（BALF）were collected for follow-up experiments.（3）Hematoxylin-eosin（HE）staining was used to observe the pathological morphology of lung injury in wild-type and Nur77 knockout mice,and the wet/dry ratio（W/D）was used to evaluate pulmonary edema.ELISA was used to detect the expression of pro-inflammatory cytokines TNF-ɑand IL-1βin lung tissue.Using Western blot,immunohistochemical detection PAD4 in lung tissue,the expression of MPO.Western blot detection Nur77,MPO,PAD4,Cit-H3 expression in lung tissue.Immunofluorescence staining detection of Cit-H3,MPO in lung tissue were expressed.Results:（1）The survival time of Nur77 knockout mice at 72 hours showed that the first death time of Nur77 knockout mice after instillation of 20mg/Kg LPS in the airway was 8hours.The mortality rate at 24 hours was 50%,and the mortality rate at 72 hours was100%.The first death time of wild-type mice after intratrachis instillation of LPS was 12 hours,which was later than that of Nur77 knockout mice.The mortality rates of 8,24 and 72 hours were 0%,37.5%and 75%,respectively,which were lower than that of Nur77 knockout mice.（2）In the Nur77-/-+NS group and WT+NS group,the lung tissue structure was intact,and no pathological damage was observed（P>0.05）.Pathological lesions were observed in the lung tissues of Nur77-/-+LPS group and WT+LPS group at 4 h,12 h and 24 h time.The pathological lesions of Nur77 knockout mice were more severe than those of wild mice,and the pathological scores were higher,with statistical significance（P<0.05）.There were no significant differences in the levels of total protein,W/D and proinflammatory factor in alveolar lavage fluid between Nur77-/-+NS group and WT+NS group（P>0.05）.After LPS treatment,Nur77 knockout mice and wild-type mice alveolar lavage total protein,W/D,proinflammatory factor level from 4 h to 24 h is obviously rising trend,and Nur77 knockout mice increased more significantly（P<0.05）.（3）using immunohistochemistry and immunofluorescence and Western blot detection ARDS NETosis expression in lung tissue of mice,Nur77-/-+NS group and WT+NS group almost did not see NETosis express.NETosis expression was similar between Nur77-/-+LPS group and WT+LPS group at 4 h（P>0.05）.The expression of NETosis in Nur77 knockout mice was higher than that in wild-type mice at 12 h and 24 h after intratairway instillation of LPS（P<0.05）.Western blot results showed that the expression of NETosis in wild-type mice was lower than that in Nur77-/-mice at 4h,12h and 24h after intratracheally instilled LPS（P<0.05）,and the expression of NETosis in Nur77 gene knockout ARDS mice was higher than that in wild-type ARDS mice.Lung injury was more severe（P<0.05）.Conclusion:Nur77 delays disease onset and mortality in severe ARDS mice;Nur77 may play an anti-inflammatory role by down-regulating the net production of NETosis to reduce the lung tissue damage in ARDS mice. |
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